The T120P or M172V mutation on rv2172c confers high level para-aminosalicylic acid resistance in Mycobacterium tuberculosis.

AbstractAlthough para-aminosalicylic acid (PAS) has been used to treat tuberculosis for decades, mechanisms of resistance to this drug in Mycobacterium tuberculosis (M. tuberculosis) clinical isolates have not been thoroughly investigated. Previously, we found that decreased methylenetetrahydrofolate reductase (MTHFR) activity of Rv2172c led to increased sensitivity to antifolates in M. tuberculosis. In this study, we collected the genome-sequencing data of 173 PAS-resistant and 803 PAS-sensitive clinical isolates and analyzed rv2172c mutations in those 976 isolates. The results showed that two mutations (T120P and M172 V) on rv2172c could be identified in a certain proportion (6.36%) of PAS-resistant isolates. The results of AlphaFold2 prediction indicated that the T120P or M172 V mutation might affect the enzymatic activity of Rv2172c by influencing nicotinamide adenine dinucleotide (NADH) binding, and this was verified by subsequent biochemical analysis, demonstrating the role of residues Thr120 and Met172 on NADH binding and enzymatic activity of Rv2172c. In addition, the effect of rv2172c T120P or M172 V mutation on methionine production and PAS resistance was determined in M. tuberculosis. The results showed that both T120P and M172 V mutations caused increased intracellular methionine concentrations and high level PAS resistance. In summary, we discovered new molecular markers and also a novel mechanism of PAS resistance in M. tuberculosis clinical isolates and broadened the understanding of the NADH-dependent MTHFR catalytic mechanism of Rv2172c in M. tuberculosis, which will facilitate the molecular diagnosis of PAS resistance and also the development of new drugs targeting Rv2172c.

Medication nonadherence and associated factors in patients with tuberculosis in Wau, South Sudan: a cross- sectional study using the world health organization multidimensional adherence model.

BACKGROUND: Tuberculosis medication nonadherence is a multi-dimensional public health problem with serious consequences worldwide. There is little information available for medication nonadherence in South Sudan. This study assessed the proportion, reasons, and associated factors for nonadherence among patients with TB in Wau Municipality, South Sudan. METHODS: A health facility based cross-sectional study was conducted among 234 tuberculosis (TB) patients receiving first line anti-TB regimen in Wau Municipality. Urine isoniazid metabolite testing (IsoScreen®) was used to determine nonadherence (visualized by negative test results) and a questionnaire was used to describe the reasons for nonadherence. Modified poisson regression with robust standard errors was performed since the proportion of nonadherence was < 10%, to identify nonadherence associated factors using the WHO Multidimensional adherence model. RESULTS: Out of 234 participants, 24.8% (95% CI, 19.2 - 30.3) were nonadherent to the TB treatment regimen. At multivariate analysis, nonadherence was significantly associated with: relief of symptoms (APR 1.93, 95% CI 1.12 - 3.34, p = 0.018), alcohol use (APR 2.12, 95% CI 1.33 - 3.96, p = 0.019) and waiting time to receive drugs (APR 1.77, 95% CI 1.11 - 2.83, p = 0.017). CONCLUSION: Tuberculosis medication nonadherence was high, and it's associated with patients' relived of symptoms, alcohol use, and prolonged waiting time at health facility. Hence, addressing these barriers and the use of multifaceted interventions e.g. counseling, health education and improve appointments are crucial to reduce nonadherence among patients with TB in South Sudan.

Sustained transmission over two decades of a previously unrecognised MPT64 negative Mycobacterium tuberculosis strain in Queensland, Australia: a whole genome sequencing study.

Background: MPT64 is a key protein used for Mycobacterium tuberculosis (MTB) complex strain identification. We describe protracted transmission of an MPT64 negative MTB strain in Queensland, Australia, and explore genomic factors related to its successful spread. Methods: All MPT64 negative strains identified between 2002 and 2022 by the Queensland Mycobacteria Reference Laboratory, and an additional 2 isolates from New South Wales (NSW), were whole genome sequenced. Bayesian modelling and phylogeographical analyses were used to assess their evolutionary history and transmission dynamics. Protein structural modelling to understand the putative functional effects of the mutated gene coding for MPT64 protein was performed. Findings: Forty-three MPT64 negative isolates were sequenced, belonging to a single MTB cluster of Lineage 4.1.1.1 strains. Combined with a UK dataset of the same lineage, molecular dating estimated 1990 (95% HPD 1987-1993) as the likely time of strain introduction into Australia. Although the strain has spread over a wide geographic area and new cases linked to the cluster continue to arise, phylodynamic analysis suggest the outbreak peaked around 2003. All MPT64 negative strains had a frame shift mutation (delAT, p.Val216fs) within the MPT64 gene, which confers two major structural rearrangements at the C-terminus of the protein. Interpretation: This study uncovered the origins of an MPT64 negative MTB outbreak in Australia, providing a richer understanding of its biology and transmission dynamics, as well as guidance for clinical diagnosis and public health action. The potential spread of MPT64 negative strains undermines the diagnostic utility of the MPT64 immunochromatographic test. Funding: This study was funded from an operational budget provided to the Queensland Mycobacterium Reference Laboratory by Pathology Queensland, Queensland Department of Health.

Regulatory role of Mycobacterium tuberculosis MtrA on dormancy/resuscitation revealed by a novel target gene-mining strategy.

Introduction: The unique dormancy of Mycobacterium tuberculosis plays a significant role in the major clinical treatment challenge of tuberculosis, such as its long treatment cycle, antibiotic resistance, immune escape, and high latent infection rate. Methods: To determine the function of MtrA, the only essential response regulator, one strategy was developed to establish its regulatory network according to high-quality genome-wide binding sites. Results and discussion: The complex modulation mechanisms were implied by the strong bias distribution of MtrA binding sites in the noncoding regions, and 32.7% of the binding sites were located inside the target genes. The functions of 288 potential MtrA target genes predicted according to 294 confirmed binding sites were highly diverse, and DNA replication and damage repair, lipid metabolism, cell wall component biosynthesis, cell wall assembly, and cell division were the predominant pathways. Among the 53 pathways shared between dormancy/resuscitation and persistence, which accounted for 81.5% and 93.0% of the total number of pathways, respectively, MtrA regulatory genes were identified not only in 73.6% of their mutual pathways, but also in 75.4% of the pathways related to dormancy/resuscitation and persistence respectively. These results suggested the pivotal roles of MtrA in regulating dormancy/resuscitation and the apparent relationship between dormancy/resuscitation and persistence. Furthermore, the finding that 32.6% of the MtrA regulons were essential in vivo and/or in vitro for M. tuberculosis provided new insight into its indispensability. The findings mentioned above indicated that MtrA is a novel promising therapeutic target for tuberculosis treatment since the crucial function of MtrA may be a point of weakness for M. tuberculosis.

Epidemiology and antimicrobial resistance of Mycobacterium spp. in the United Arab Emirates: a retrospective analysis of 12 years of national antimicrobial resistance surveillance data.

Introduction: The Eastern Mediterranean Regional Office (EMRO) region accounts for almost 8% of all global Mycobacterium tuberculosis (TB) cases, with TB incidence rates ranging from 1 per 100,000 per year in the United Arab Emirates (UAE) to 204 per 100,000 in Djibouti. The national surveillance data from the Middle East and North Africa (MENA) region on the epidemiology and antimicrobial resistance trends of TB, including MDR-TB remains scarce. Methods: A retrospective 12-year analysis of N = 8,086 non-duplicate diagnostic Mycobacterium tuberculosis complex (MTB complex) isolates from the UAE was conducted. Data were generated through routine patient care during the 2010-2021 years, collected by trained personnel and reported by participating surveillance sites to the UAE National Antimicrobial Resistance (AMR) Surveillance program. Data analysis was conducted with WHONET, a windows-based microbiology laboratory database management software developed by the World Health Organization Collaborating Center for Surveillance of Antimicrobial Resistance, Boston, United States (https://whonet.org/). Results: A total of 8,086 MTB-complex isolates were analyzed. MTB-complex was primarily isolated from respiratory samples (sputum 80.1%, broncho-alveolar lavage 4.6%, pleural fluid 4.1%). Inpatients accounted for 63.2%, including 1.3% from ICU. Nationality was known for 84.3% of patients, including 3.8% Emiratis. Of UAE non-nationals, 80.5% were from 110 countries, most of which were Asian countries. India accounted for 20.8%, Pakistan 13.6%, Philippines 12.7%, and Bangladesh 7.8%. Rifampicin-resistant MTB-complex isolates (RR-TB) were found in 2.8% of the isolates, resistance to isoniazid, streptomycin, pyrazinamide, and ethambutol, was 8.9, 6.9, 3.4 and 0.4%, respectively. A slightly increasing trend of resistance among MTB-complex was observed for rifampicin from 2.5% (2010) to 2.8% (2021). Conclusion: Infections due to MTB-complex are relatively uncommon in the United Arab Emirates compared to other countries in the MENA region. Most TB patients in the UAE are of Asian origin, mainly from countries with a high prevalence of TB. Resistance to first line anti-tuberculous drugs is generally low, however increasing trends for MDR-TB mainly rifampicin linked resistance is a major concern. MDR-TB was not associated with a higher mortality, admission to ICU, or increased length of hospitalization as compared to non-MDR-TB.

Culture-Free Whole Genome Sequencing of Mycobacterium tuberculosis Using Ligand-Mediated Bead Enrichment Method.

Background: Direct whole genome sequencing (WGS) of Mycobacterium tuberculosis (Mtb) can be used as a tool to study drug resistance, mixed infections, and within-host diversity. However, WGS is challenging to obtain from clinical samples due to low number of bacilli against a high background. Methods: We prospectively collected 34 samples (sputum, n = 17; bronchoalveolar lavage, n = 13; and pus, n = 4) from patients with active tuberculosis (TB). Prior to DNA extraction, we used a ligand-mediated magnetic bead method to enrich Mtb from clinical samples and performed WGS on Illumina platform. Results: Mtb was definitively identified based on WGS from 88.2% (30/34) of the samples, of which 35.3% (12/34) were smear negative. The overall median genome coverage was 15.2% (interquartile range [IQR], 7.7%-28.2%). There was a positive correlation between load of bacilli on smears and genome coverage (P < .001). We detected 58 genes listed in the World Health Organization mutation catalogue in each positive sample (median coverage, 85% [IQR, 61%-94%]), enabling the identification of mutations missed by routine diagnostics. Mutations causing resistance to rifampicin, isoniazid, streptomycin, and ethambutol were detected in 5 of 34 (14.7%) samples, including the rpoB S441A mutation that confers resistance to rifampicin, which is not covered by Xpert MTB/RIF. Conclusions: We demonstrate the feasibility of magnetic bead-based enrichment for culture-free WGS of Mtb from clinical specimens, including smear-negative samples. This approach can also be integrated with low-cost sequencing workflows such as targeted sequencing for rapid detection of Mtb and drug resistance.

Overexpression of LAG-3: a potential indicator of low immune function in tuberculosis.

Background: Tuberculosis (TB) persists as a global health challenge, with its treatment hampered by the side effects of long-term combination drug therapies and the growing issue of drug resistance. Therefore, the development of novel therapeutic strategies is critical. This study focuses on the role of immune checkpoint molecules (ICs) and functions of CD8+ T cells in the search for new potential targets against TB. Methods: We conducted differential expression genes analysis and CD8+ T cell functional gene analysis on 92 TB samples and 61 healthy individual (HI) samples from TB database GSE83456, which contains data on 34,603 genes. The GSE54992 dataset was used to validated the findings. Additionally, a cluster analysis on single-cell data from primates infected with mycobacterium tuberculosis and those vaccinated with BCG was performed. Results: The overexpression of LAG-3 gene was found as a potentially important characteristic of both pulmonary TB (PTB) and extrapulmonary TB (EPTB). Further correlation analysis showed that LAG-3 gene was correlated with GZMB, perforin, IL-2 and IL-12. A significant temporal and spatial variation in LAG-3 expression was observed in T cells and macrophages during TB infection and after BCG vaccination. Conclusion: LAG-3 was overexpressed in TB samples. Targeting LAG-3 may represent a potential therapeutic target for tuberculosis.

Paradoxical Reaction in Intraocular Tuberculosis: Report of Three Cases.

PURPOSE: To present paradoxical reaction (PR) in three cases with ocular tuberculosis (OTB) treated with antitubercular therapy (ATT), highlighting diagnostic challenges and treatment strategies. METHODS: We retrospectively reviewed clinical records of three OTB patients presenting with paradoxical worsening after ATT initiation at two Brazilian university hospitals. RESULTS: The patients (2 males, 1 female) experienced worsening clinical presentation (increased inflammation, vision loss) within two to three weeks after initiating ATT. One patient who was HIV-positive with unilateral multifocal choroiditis developed PR soon after starting antiretroviral therapy. The second patient presented with a choroidal tuberculoma in both eyes. The third patient also had multifocal choroiditis and developed a localized choroidal elevation with a double-layer sign as a manifestation of PR. All patients were maintained on ATT therapy in association with corticosteroids and experienced improvement of inflammatory signs. CONCLUSION: This case series highlights the potential for PR in OTB patients. Close monitoring and prompt therapeutic adjustments are crucial for management success.

The anti-tubercular callyaerins target the Mycobacterium tuberculosis-specific non-essential membrane protein Rv2113.

Spread of antimicrobial resistances urges a need for new drugs against Mycobacterium tuberculosis (Mtb) with mechanisms differing from current antibiotics. Previously, callyaerins were identified as promising anti-tubercular agents, representing a class of hydrophobic cyclopeptides with an unusual (Z)-2,3-di-aminoacrylamide unit. Here, we investigated the molecular mechanisms underlying their antimycobacterial properties. Structure-activity relationship studies enabled the identification of structural determinants relevant for antibacterial activity. Callyaerins are bacteriostatics selectively active against Mtb, including extensively drug-resistant strains, with minimal cytotoxicity against human cells and promising intracellular activity. By combining mutant screens and various chemical proteomics approaches, we showed that callyaerins target the non-essential, Mtb-specific membrane protein Rv2113, triggering a complex dysregulation of the proteome, characterized by global downregulation of lipid biosynthesis, cell division, DNA repair, and replication. Our study thus identifies Rv2113 as a previously undescribed Mtb-specific drug target and demonstrates that also non-essential proteins may represent efficacious targets for antimycobacterial drugs.

Retrograde Transvenous Thoracic Duct Embolization for Tubercular Chylothorax.

This case study documents the clinical profile of a 27-year-old male patient who visited the medical facility two months ago with complaints of dry cough, fatigue, weight loss, and occasional fever. He had been treated for ascites and pleural effusion in the hospital before presentation and returned with an intercostal drain in place. A detailed examination revealed symptoms of respiratory disorders, including fluid in both lungs, fever, and dyspnea. His fluid levels showed multiple deviations from the normal range, according to the report's findings and lab test results. It was determined that the patient had chylothorax, which resulted from hemophagocytic lymphohistiocytosis (HLH) and abdominal tubercular lymphadenopathy. His anti-tubercular treatment (AKT4) was initiated, along with octreotide for his management. Initial management included non-invasive ventilator (NIV) support, intravenous antibiotics, nebulization, and an intercostal chest drain (ICD). Later, the patient underwent retrograde transvenous thoracic duct embolization (TDE) using N-butyl cyanoacrylate (NBCA) glue. The removal of the drainage tube and the patient's stable discharge were made possible through regular monitoring and collaboration between specialists.

Epigenetic modulation of cytokine expression in Mycobacterium tuberculosis-infected monocyte derived-dendritic cells: Implications for tuberculosis diagnosis.

BACKGROUND: To delineate alterations in DNA methylation at high resolution within the genomic profile of monocyte-derived-dendritic cells (mo-DCs) in connection with Mycobacterium tuberculosis (MTB) infection, with particular emphasis on pro/ anti-inflammatory genes. METHODS: In the context of this investigation, mo-DCs were infected by various active strains of MTB (Rifampicin-resistant [RIFR], H37Rv, multidrug-resistant [MDR], and extensively drug-resistant [XDR]). Subsequently, the pro/anti-inflammatory hub gene expression levels within the IL-6, IL-12, IFN-γ, IL-1ß, TNF-α, and IL-10 pathways were evaluated employing real-time reverse transcription-polymerase chain reaction (RT-PCR). Additionally, the effects of MTB infection on mo-DC protein expression were examined through western blot analysis. The methylation status (%) of TNF-α and IL-10 was considered through Methylation Sensitive-High Resolution Melting (MS-HRM). RESULTS: The results revealed an up-regulation of all pro-inflammatory genes among all groups, with TNF-α exhibiting the highest expression level. Conversely, the anti-inflammatory gene (IL-10) showed a down-regulated expression level. Furthermore, the DNA methylation status (%) of TNF-α decreased significantly among all the groups (P < 0.001), although there were no notable distinctions in the DNA methylation status (%) of IL-10 when compared to the control group (P > 0.05). CONCLUSION: MTB infection induces DNA methylation changes in mo-DCs. The hypo-methylation of TNF-α may induce the up-regulation of this gene. This correlation revealed that the more resistant the MTB strain (XDR) is, the lower the methylation status (%) in the TNF-α gene.

Physiologic medium renders human iPSC-derived macrophages permissive for M. tuberculosis by rewiring organelle function and metabolism.

In vitro studies are crucial for our understanding of the human macrophage immune functions. However, traditional in vitro culture media poorly reflect the metabolic composition of blood, potentially affecting the outcomes of these studies. Here, we analyzed the impact of a physiological medium on human induced pluripotent stem cell (iPSC)-derived macrophages (iPSDM) function. Macrophages cultured in a human plasma-like medium (HPLM) were more permissive to Mycobacterium tuberculosis (Mtb) replication and showed decreased lipid metabolism with increased metabolic polarization. Functionally, we discovered that HPLM-differentiated macrophages showed different metabolic organelle content and activity. Specifically, HPLM-differentiated macrophages displayed reduced lipid droplet and peroxisome content, increased lysosomal proteolytic activity, and increased mitochondrial activity and dynamics. Inhibiting or inducing lipid droplet formation revealed that lipid droplet content is a key factor influencing macrophage permissiveness to Mtb. These findings underscore the importance of using physiologically relevant media in vitro for accurately studying human macrophage function. IMPORTANCE: This work compellingly demonstrates that the choice of culture medium significantly influences M. tuberculosis replication outcomes, thus emphasizing the importance of employing physiologically relevant media for accurate in vitro host-pathogen interaction studies. We anticipate that our work will set a precedent for future research with clinical relevance, particularly in evaluating antibiotic efficacy and resistance in cellulo.

Gingiva as the primary site of extrapulmonary tuberculosis: A rare case report with brief review of literature.

Multiple strains of Mycobacteria cause tuberculosis (TB), a chronic, specific infectious granulomatous disease. It mainly occurs with pulmonary involvement when compared to extrapulmonary involvement. Primary oral occurrence is uncommon and oral lesions are usually secondary to pulmonary involvement. When there are no active pulmonary clinical manifestations of TB, the diagnosis of the very rare entity of primary gingival TB poses a great challenge to clinicians. In this case report, we discuss a case of primary gingival TB in a 24-year-old lactating mother. This article briefs the onset and course of the lesion during pregnancy and postpartum, elaborates the pathway to diagnosis, various investigations performed and the regimen of antitubercular therapy for 6 months, followed by complete resolution of the lesion without recurrence. This report also describes the significance of considering TB as a differential diagnosis in oral lesions and the various diagnostic methods available. It also emphasizes the sole importance of histopathology in the early detection of the lesion and its management.

Retrospective analysis of the clinical utility of multi-cytokine profiles in smear-negative pulmonary tuberculosis.

OBJECTIVES: To evaluate cytokine profiles and interferon-gamma release assay (IGRA) for their diagnostic capabilities in the differentiation of tuberculosis (TB) from non-TB conditions, as well as smear-negative pulmonary tuberculosis (SNPT) from smear-positive pulmonary tuberculosis (SPPT). METHODS: A total of 125 participants were included, 77 of whom had TB and 48 who didn't, and demographic, clinical, and laboratory data were collected, including cytokine levels and IGRA results. The TB patients were further divided into 2 subgroups: SNPT (n=42) and SPPT (n=35). RESULTS: Compared to non-TB, the TB group had lower BMI, higher WBC, neutrophils, monocytes, ESR and CRP (p<0.05). TB patients showed higher IL-2, IL-6, IFN-γ, IL-8 (p<0.001) and higher IGRA positivity (88.3% versus [vs.] 29.2%, p<0.001). Between SNPT and SPPT, moderate effect sizes were observed for IFN-α, IL-2, IL-10, IL-8 (Cohen's d 0.59-0.76), with lower IGRA positivity in SNPT (81.0% vs. 97.1%, p=0.015). ROC analysis indicated IFN-α, IL-2, IL-10, IL-8 had moderate accuracy for SNPT diagnosis (AUCs 0.668-0.734), and combining these improved accuracy (AUC 0.759, 80% sensitivity, 64.2% specificity). CONCLUSION: A multi-biomarker approach combining these cytokines demonstrates enhanced diagnostic accuracy for tuberculosis.

Immunoinformatics and structural aided approach to develop multi-epitope based subunit vaccine against Mycobacterium tuberculosis.

Tuberculosis is a highly contagious disease caused by Mycobacterium tuberculosis (Mtb), which is one of the prominent reasons for the death of millions worldwide. The bacterium has a substantially higher mortality rate than other bacterial diseases, and the rapid rise of drug-resistant strains only makes the situation more concerning. Currently, the only licensed vaccine BCG (Bacillus Calmette-Guérin) is ineffective in preventing adult pulmonary tuberculosis prophylaxis and latent tuberculosis re-activation. Therefore, there is a pressing need to find novel and safe vaccines that provide robust immune defense and have various applications. Vaccines that combine epitopes from multiple candidate proteins have been shown to boost immunity against Mtb infection. This study applies an immunoinformatic strategy to generate an adequate multi-epitope immunization against Mtb employing five antigenic proteins. Potential B-cell, cytotoxic T lymphocyte, and helper T lymphocyte epitopes were speculated from the intended proteins and coupled with 50 s ribosomal L7/L12 adjuvant, and the vaccine was constructed. The vaccine's physicochemical profile demonstrates antigenic, soluble, and non-allergic. In the meantime, docking, molecular dynamics simulations, and essential dynamics analysis revealed that the multi-epitope vaccine structure interacted strongly with Toll-like receptors (TLR2 and TLR3). MM-PBSA analysis was performed to ascertain the system's intermolecular binding free energies accurately. The immune simulation was applied to the vaccine to forecast its immunogenic profile. Finally, in silico cloning was used to validate the vaccine's efficacy. The immunoinformatics analysis suggests the multi-epitope vaccine could induce specific immune responses, making it a potential candidate against Mtb. However, validation through the in-vivo study of the developed vaccine is essential to assess its efficacy and immunogenicity profile, which will assure active protection against Mtb.

Anti-tuberculosis effect of microbiome therapeutic PMC205 in extensively drug-resistant pulmonary tuberculosis in vivo.

BACKGROUND: Tuberculosis is a highly contagious disease caused by Mycobacterium tuberculosis, and the increase in antibiotic resistance threatens humankind. Therefore, there is an urgent need to develop new anti-tuberculosis drugs that can overcome the limitations of existing drugs. Here, we report the anti-tuberculosis effect of microbiome therapeutic PMC205, a strain of Bacillus subtilis. METHODS: The anti-tuberculosis activity of probiotics was evaluated in mouse models of lethal and latent pulmonary tuberculosis induced by high or low-dose infection of the extensively drug-resistant (XDR) strain. Probiotics were administered by inhalation, and the burden of M. tuberculosis in the lungs, along with mortality and clinical observations, were monitored for 12 weeks and 8 months, respectively. For an in-depth understanding, analysis of the microbiome and inflammatory profile of the lung microenvironment and induction of autophagy in vitro were explored. RESULTS: After inhalation administration of PMC205 for 3 months, the survival rate was 100%, unlike all deaths in the saline-treated group, and the burden of M. tuberculosis in the lungs was reduced by log 1.3 in the 8-month latent tuberculosis model. Moreover, PMC205 induced recovery of disrupted lung microflora, increased butyric acid, and suppressed excessive inflammation. It also promoted autophagy. CONCLUSIONS: These results confirm PMC205's anti-tuberculosis effect, suggesting that it can be developed as an adjuvant to current antibiotic therapy to solve the drug-resistant tuberculosis problem.

Imidazoquinoline Derivatives as Potential Inhibitors of InhA Enzyme and Mycobacterium tuberculosis.

Tuberculosis is a serious public health problem worldwide. The search for new antibiotics has become a priority, especially with the emergence of resistant strains. A new family of imidazoquinoline derivatives, structurally analogous to triazolophthalazines, which had previously shown good antituberculosis activity, were designed to inhibit InhA, an essential enzyme for Mycobacterium tuberculosis survival. Over twenty molecules were synthesized and the results showed modest inhibitory efficacy against the protein. Docking experiments were carried out to show how these molecules could interact with the protein's substrate binding site. Disappointingly, unlike triazolophthlazines, these imidazoquinoline derivatives showed an absence of inhibition on mycobacterial growth.

The Influence of Body Fat Dynamics on Pulmonary Immune Responses in Murine Tuberculosis: Unraveling Sex-Specific Insights.

The World Health Organization (WHO) highlights a greater susceptibility of males to tuberculosis (TB), a vulnerability attributed to sex-specific variations in body fat and dietary factors. Our study delves into the unexplored terrain of how alterations in body fat influence Mycobacterium tuberculosis (Mtb) burden, lung pathology, immune responses, and gene expression, with a focus on sex-specific dynamics. Utilizing a low-dose Mtb-HN878 clinical strain infection model, we employ transgenic FAT-ATTAC mice with modulable body fat to explore the impact of fat loss (via fat ablation) and fat gain (via a medium-fat diet, MFD). Firstly, our investigation unveils that Mtb infection triggers severe pulmonary pathology in males, marked by shifts in metabolic signaling involving heightened lipid hydrolysis and proinflammatory signaling driven by IL-6 and localized pro-inflammatory CD8+ cells. This stands in stark contrast to females on a control regular diet (RD). Secondly, our findings indicate that both fat loss and fat gain in males lead to significantly elevated (1.6-fold (p ≤ 0.01) and 1.7-fold (p ≤ 0.001), respectively) Mtb burden in the lungs compared to females during Mtb infection (where fat loss and gain did not alter Mtb load in the lungs). This upsurge is associated with impaired lung lipid metabolism and intensified mitochondrial oxidative phosphorylation-regulated activity in lung CD8+ cells during Mtb infection. Additionally, our research brings to light that females exhibit a more robust systemic IFNγ (p ≤ 0.001) response than males during Mtb infection. This heightened response may either prevent active disease or contribute to latency in females during Mtb infection. In summary, our comprehensive analysis of the interplay between body fat changes and sex bias in Mtb infection reveals that alterations in body fat critically impact pulmonary pathology in males. Specifically, these changes significantly reduce the levels of pulmonary CD8+ T-cells and increase the Mtb burden in the lungs compared to females. The reduction in CD8+ cells in males is linked to an increase in mitochondrial oxidative phosphorylation and a decrease in TNFα, which are essential for CD8+ cell activation.

Optimization of Mycobacterium tuberculosis DNA processing prior to whole genome sequencing.

The process of whole genome sequencing of the Mycobacterium tuberculosis complex is dependent on complete the inactivation of the strain and subsequent DNA extraction. The objective of this study was to optimise the two steps. Firstly, the efficacy of Triton X-100 as a solvent for the inactivation step was evaluated. This solvent has been demonstrated to be effective in killing bacteria and is less toxic than the previously employed chloroform. For the extraction step, two lysis methods were evaluated: enzymatic (B1 protocol) and mechanical (B2 protocol). For whole genome sequencing, the Nextera XT DNA library preparation protocol was performed for both the B1 and B2 protocols. Subsequently, each library was subjected to whole-genome sequencing. The results demonstrated that heat lysis inactivation with Triton was effective, with no bacteria remaining viable following this treatment. The enzymatic and mechanical extraction protocols yielded comparable results in terms of DNA quantity and quality. The sequencing results showed that there was no significant difference in read depths between the two protocols. In conclusion, for MTBC strains, we recommend the use of our Triton inactivation method, which meets biosafety expectations.

Immunogenic profiling of Mycobacterium tuberculosis Rv1513 reveals its ability to switch on Th1 based immunity.

Tuberculosis (TB) is one of the infectious diseases caused by the pathogen Mycobacterium tuberculosis that continuously threatens the global human health. Bacillus Calmette-Guérin (BCG) vaccine is the only vaccine that has been used clinically to prevent tuberculosis in recent centuries, but its limitations in preventing latent infection and reactivation of tuberculosis do not provide full protection. In this study, we selected the membrane-associated antigen Rv1513 of Mycobacterium. In order to achieve stable expression and function of the target gene, the prokaryotic expression recombinant vector pET30b-Rv1513 was constructed and expressed and purified its protein. Detection of IFN- γ levels in the peripheral blood of TB patients stimulated by whole blood interferon release assay (WBIA) and multi-microsphere flow immunofluorescence luminescence (MFCIA) revealed that the induced production of cytokines, such as IFN-γ and IL-6, was significantly higher than that in the healthy group. Rv1513 combined with adjuvant DMT (adjuvant system liposomes containing dimethyldioctadecylammonium bromide (DDA), monophospholipid A (MPL), and trehalose-660-dibenzoic acid (TDB)) was used to detect serum specific antibodies, cytokine secretion from splenic suprasplenic cell supernatants, and multifunctional T-cell levels in splenocytes in immunised mice. The levels of IFN-γ, TNF-α, and IL-2 secreted by mouse splenocytes were found in the Rv1513+DMT group and the BCG+Rv1513+DMT group. The serum levels of IgG and its subclasses and the number of IFN-γ+T cells, TNF-α+T and IFN-γ+TNF-α+T cells in the induced CD4+/CD8+T cells in mice were significantly higher than those in the BCG group, and the highest levels were found in the BCG+Rv1513+DMT group. These findings suggest that Rv1513/DMT may serve as a potential subunit vaccine candidate that may be effective as a booster vaccine after the first BCG vaccination.